Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile.

Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a highly contagious disease that is endemic to salmon farming in Chile and causes great economic losses to the industry. Here we compared different diagnostic methods to detect IPNV in field samples, including 3 real-time reverse transcription PCR (qRT-PCR) assays, cell culture isolation, and indirect fluorescent antibody test (IFAT). Additionally, we performed a phylogenetic analysis to investigate the genogroups prevailing in Chile, as well as their geographic distribution and virulence. The 3 qRT-PCR assays used primers that targeted regions of the VP2 and VP1 genes of the virus and were tested in 46 samples, presenting a fair agreement within their results. All samples were positive for at least 2 of the qRT-PCR assays, 29 were positive for cell culture, and 23 for IFAT, showing less sensitivity for these latter 2 methods. For the phylogenetic analysis, portions of 1180 and 523 bp of the VP2 region of segment A were amplified by RT-PCR, sequenced and compared with sequences from reference strains and from isolates reported by previous studies carried out in Chile. Most of the sequenced isolates belonged to genogroup 5 (European origin), and 5 were classified within genogroup 1 (American origin). Chilean isolates formed clusters within each of the genogroups found, evidencing a clear differentiation from the reference strains. To our knowledge, this is the most extensive study completed for IPNV in Chile, covering isolates from sea- and freshwater salmon farms and showing a high prevalence of this virus in the country.

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins.

The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.

Infectious pancreatic necrosis virus (IPNV) does not require acid compartments for entry into cells.

The early events in the infection of unenveloped viruses are still rather unknown and puzzling. However, as in the case of enveloped viruses, the acid pH of endosomes can be important to trigger the virus entry into the cytosol. To test if the infectious pancreatic necrosis virus (IPNV), an unenveloped virus, requires acid endosomal pH to infect cells, we assayed the effect of Bafilomycin A1 on IPNV replication. Concentrations of the antibiotic which inhibited the endosomal acidification of the host cells were unable to affect IPNV replication in CHSE-214 cells; therefore, the acid pH of endosomes seems not to be a mandatory condition for the entry of IPNV into cells.

Attachment and entry of infectious pancreatic necrosis virus (IPNV) into CHSE-214 cells.

Infectious pancreatic necrosis virus (IPNV) attaches to CHSE-214 cells through two types of cell components: specific and non-specific ones. Competition experiments with inactivated IPNV showed that IPNV requires specific components to productively infect cells. Just a low amount of adsorbed IPNV enters the cell. After 20 minutes, part of the adsorbed IPNV was internalized into acid compartments. Also, the viruses adsorbed on the cell surface require similar periods of time to escape from the neutralization of antibodies.

Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific infectivity.

An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described. Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1. IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl. The bovine serum albumin content of the growth medium supplement also elutes close to this position. Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).

Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.

Further analysis on the structural proteins of infectious pancreatic necrosis virus.

The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.

Effect of ammonium chloride on the multiplication of infectious pancreatic necrosis virus.

The multiplication of IPNV in CHSE cells is inhibited by ammonium chloride. This inhibition is complete if NH4Cl is added early in the infective cycle. Immunofluorescence analysis shows that NH4Cl reduces the number of virus producing cells and thus suggests it may act by inhibiting virus internalization. In addition, NH4Cl can produce partial inhibition of IPNV multiplication if added later in the replicative cycle. Analysis of 3H-uridine incorporation into viral RNA shows that this later effect could be an inhibition of viral RNA synthesis.

Identity between infectious pancreatic necrosis virus VR-299 and a Chilean isolate.

Nucleic acid and protein electrophoretic analyses of the Chilean isolate of infectious pancreatic necrosis virus showed that genomic RNA, as well as the major proteins, were indistinguishable from those of the VR-299 serotype. The data are in good agreement with the theory that the virus was introduced into Chile from North America.

Effect of rifamycin derivatives and coumermycin A1 on in vitro RNA synthesis by African swine fever virus. Brief report.

Several rifamycin derivatives inhibited the DNA-dependent RNA polymerase of African swine fever (ASF) virus particles. The inhibition was similar to that found with vaccinia virus RNA polymerase. Coumermycin A1, an inhibitor of type II DNA topoisomerases, inhibited strongly RNA synthesis in vitro by ASF virus particles. This suggests that transcription of ASF virus DNA requires a DNA topoisomerase.

Nucleoside triphosphate phosphohydrolase activities in African swine fever virus.

African swine fever virus contains nucleoside triphosphate phosphohydrolase activity which releases 32P phosphate from gamma-32P ATP at a rate of about 1 mumol/h mg of virus protein. The hydrolase activity is slightly stimulated by adding nucleic acids to the reaction mixture and under conditions of RNA synthesis. A study of the rate of ATP hydrolysis at different concentrations of ATP suggests the existence of two phosphohydrolase activities with apparent Km values of about 0.04 and 1 mM.